Study On The Methods Of Real Time Immuno-Polymerase Chain Reaction For Polychlorinated Biphenyls

"Environmental hormones" is a term given to endocrine disruptors, which are chemical substances capable of disordering animal hormones. Hence, it adversely affects the endocrine system and consequently alters the development and/or reproduction of organisms, including humans. Polychlorinated biphenyls (PCBs) are routinely used as additives in various oil-based preparations and are important in the manufacture of products as diverse as plastics and pesticides. The toxicological study indicates that the PCBs mimic, impair, enhance or inhibit the actions of endogenous hormones, which cause malignant tumors and abnormalities in growth and reproduction.Today, the PCBs presenting in the environment mainly root in the leftover in last century, and still partly sourced from nature and anthropogenic processes. Due to the stability of chemical structure, the PCBs are uneasily removed by the sunlight and microbe. Therefore the PCBs distribute worldwide in the environment and become one of the most prevalent pollutants. It has been banned in industrialized countries and monitored routinely as priority pollutant for regulatory purposes, and the stringent discharge standards are issued to control the new PCBs in the environment.Gas chromatography, high performance liquid chromatography and high-resolution gas chromatography/mass spectrometry (HGGC/HGMS) are the usual methods for determination of PCBs. However there are some shortcomings in these methods, such as expensive instruments, professional techniques, infeasibility of the on-site analyses and batch testing in practice samples. In contrast, immunoassays are typically very sensitive and readily adapted to analysis for which an appropriate antibody is available. Nowadays, there were many reliable immune methods in detecting PCBs, such as ELISAs, biosensor and SPR. In this paper, we will use a new immunoassay for detecting PCBs in the environment.The IPCR method, first described by Sano et al. in 1992, combines the well-established ELISA methodology with the signal amplification power of the PCR. A number of research applications describe the advantages of the method, that is, in particular, its high sensitivity and good quantification capabilities due to the great linearity and compatibility with established ELISA protocols. Further development in the technology and instrumentation used for the signal detection of IPCR has resulted in the development of real-time IPCR (rt-IPCR).Real-time immuno-polymerase chain reaction (rt-IPCR) is a versatile and robust technique based on the advantage of rt-PCR and IPCR for the ultra-sensitive analysis of pathological proteins and other viral antigens. With its development, the method has been applied to detect antigens associated with cancer, autoimmune diseases, pathogenic bacteria and bacterial toxin in clinical diagnosis and biochemical analysis fields. But no environmental pollutants have been detected by rt-IPCR. The method’s sensitivity was higher over conventional ELISA according to the reports, and inter-assay error was lower than IPCR. This paper was to introduce this versatile technique into environmental monitoring field and set up a series of new immunoassays for determination of trace environmental hormone.In this work, three kinds of representative PCBs congeners were selected and modified for the immunoassay study. We designed and prepared optimum haptens acting as immunogens and competitors, and then characterized the artificial antigen and antibody. A series of rt-IPCR immunoassays were developed to detect PCBs in real environmental samples. The work is summarized as the following:1. Study on the methods of synthesis PCBs congeners. One PCB15 derivatives was synthesized by the method of deoxidized biphenyl amine, and three congeners were synthesized by the modified Gomberg-Bachmann reaction. All PCB congeners were characterized by IR, ~1HNMR and element analysis. A new synthesis PCB congener method was developed.2. Three PCB congener haptens were synthesized by the Friedel-crafts acylation reaction and a carboxyl was inducted to the PCB( PCB12,PCB37,PCB77) molecule. And the products were characterized by IR, ~1HNMR and element analysis technology.3. Synthesis of artificial antigens. The haptens were conjugated to the carrier proteins (BSA, OVA) with the modified active ester method or with the mixed acid anhydride method to form immune antigens and coating antigens. In this way the artificial antigen of PCBs were obtained. Structures of the products were characterized by UV spectra and detecting protein contentration.4. Preparation of polyclonal antibodies. Male New Zealand white rabbits were immunized with the mixture of immune antigens and freund adjuvant. The antiserum from the rabbit was purified by the method of octanoic acid ammonium sulfate two-step precipitation, Sephadex G-25 and DEAE cellulose. In this way the specific and affinitive pAbs of PCBs had been yielded. The sera were tested for titer by the agar diffusion test and the tube agglutination reaction test, the titer of the antibody for the PCB12,37,77 is 1:32,1:32,1:64 respectively.5. Preparation and purification of biotinylated reporter DNA:The reporter DNA with biotin is a 103-base pair sequence from the pUC19 vectors. The reporter DNA was generated by PCR as follows. The forward primer, M13/pUC sequencing as G TAA AAC GAC GGC CAG T, was biotinylated at the 5’ end to generate a biotiny group to the DNA. The reverse primer, M13/pUC reverse sequencing as CAG GAA ACA GCT ATG AC was also biotinylated at the 5’ end to generate a biotiny group to the DNA. All regents were added to PCR tube as described by the DNA PCR kit handbook. The PCR conditions were: hold 94℃for 4 min; 30 cycles of 94℃for 20 s, 55℃for 20 s, and 72℃for 20 s. The 72℃step is extended to 3 min in the final cycle. The reporter DNA was purified and retrieved by UNIQ-10 PCR DNA extraction kit. The DNA was quantified by UV absorbency and checked qualitatively using agarose gel.6. Preparation of biotinylated polyclonal antibodies and haptens. The specific and affinitive pAbs yielded in our own laboratory were biotinylated, using biotinamido-caproate-N-hydroxysuccinimide ester (BNHS) as reported. The biotinylated antibody was then purified by the semi permeable membrane dialyzing. The biotinylated haptens were synthesized by the mixed acid anhydride method using the three PCB haptens with BNHS. Structures of the products were characterized by UV spectra and detecting protein content ration.7. Indirect competitive real-time Immuno-PCR (rt-IPCR) assay for the determination of PCBs in environment was developed. Coating antigens adsorbed to the PCR tubes treated with glutaraldehyde, was used to compete with the PCBs in combining pAbs. Biotinylated goat anti-rabbit IgG were added to combine pAbs. Avidin was used as a bridge between the biotinylated goat anti-rabbit IgG and the biotinylated 103 bp reporter DNA. The reporter DNA was amplified and measured by rt-PCR under the optimize procedure. The correlative coefficients of 0.978, 0.961, 0.954 were obtained according to the PCB12, PCB 37, PCB 77.8. Direct competitive real-time Immuno-PCR (rt-IPCR) assay for the determination of PCBs in environment was developed. Coating antigens adsorbed to the PCR tubes treated with glutaraldehyde, was used to competing with the PCBs in combining biotinylated pAbs. Avidin was used as a bridge between the biotinylated goat anti-rabbit IgG and the biotinylated reporter DNA. The reporter DNA was amplified and measured by rt-PCR under the optimize procedure. The correlative coefficients of 0.987, 0.988, 0.985were obtained according to the PCB12, PCB 37, PCB 77.9. Antibody-coated real-time Immuno-PCR (rt-IPCR) assay for the determination of PCBs in environment was developed. Glutaraldehyde treated PCR tubes were adsorbed by coating pAbs specific for PCBs, with which biotinylated PCBs hapten and antigen (Ag) PCBs were competing in combining. Avidin was used as a bridge between the biotinylated PCBs hapten and the biotinylated reporter DNA. The reporter DNA was amplified and measured by rt-PCR under the optimize procedure. The correlative coefficients of 0.966, 0.971 , 0.985 were obtained according to the PCB12, PCB 37, PCB 77.Comparing the results of the three methods, we will find that the direct competitive rt-IPCR and antibody-coated rt-IPCR has better performance than the indirect competitive rt-IPCR. The reagent preparation and the assay characterization. optimization and validation steps are described. The optimum experimental situation is systematically studied with coating medium and time, the concentration of antibody, biotinylated DNA and haptens, et al respectively. After PCR tubes treated with 0.8% glutaraldehyde, the increasing absorb capability of tubes can improve the veracity of the assay. The coating medium and time are investigated, the results show that the pH 9.6 CBS is optimal coating solvent, the coating time is suitably in 4°C throughout the night, and the 3% OVA was the optimal blocking solvent. Cross-reaCtivity, recovery rates and detection limit are also observed. The results show that the cross-reactivity of the rt-IPCR is less than 15%. Good recoveries (85 - 110%) are obtained for spiked purified soil extracts. The analytical performance of the assay is consistent with GC/MS. These data indicate that the rt-IPCR is suitable for screening PCBs in environment, and there is no reports about these methods that have been detected.